ABSTRACT
G-quadruplex DNA structures (G4) are proven to interfere with most genetic and epigenetic processes. Small molecules binding these structures (G4 ligands) are invaluable tools to probe G4-biology and address G4-druggability in various diseases (cancer, viral infections). However, the large number of reported G4 ligands (>1000) could lead to confusion while selecting one for a given application. Herein we conducted a systematic affinity ranking of 11â popular G4 ligands vs 5â classical G4 sequences using FRET-melting, G4-FID assays and SPR. Interestingly SPR data globally align with the rankings obtained from the two semi-quantitative assays despite discrepancies due to limits and characteristics of each assay. In the whole, PhenDC3 emerges as the most potent binder irrespective of the G4 sequence. Immediately below PDS, PDC-360A, BRACO19, TMPyP4 and RHPS4 feature strong to medium binding again with poor G4 topology discrimination. More strikingly, the G4 drugs Quarfloxin, CX5461 and c-PDS exhibit weak affinity with all G4s studied. Finally, NMM and Cu-ttpy showed heterogeneous behaviors due, in part, to their physicochemical particularities poorly compatible with screening conditions. The remarkable properties of PhenDC3 led us to propose its use for benchmarking FRET-melting and G4-FID assays for rapid G4-affinity evaluation of newly developed ligands.
Subject(s)
G-Quadruplexes , Ligands , Humans , Fluorescence Resonance Energy Transfer , DNA/chemistry , DNA/metabolism , Surface Plasmon Resonance , Binding Sites , Molecular StructureABSTRACT
We show that nucleotides which are modified with a G-quadruplex-derived DNAzyme are substrates for DNA polymerases. Based on this finding we developed a naked-eye detection system that allows the detection of single nucleotide variations in DNA.
Subject(s)
DNA, Catalytic/chemistry , DNA, Catalytic/metabolism , DNA-Directed DNA Polymerase/metabolism , G-Quadruplexes , Nucleotides/metabolism , Benzothiazoles/chemistry , Biocatalysis , Biosensing Techniques , Models, Molecular , Oxidation-Reduction , Polymorphism, Single Nucleotide , Sulfonic Acids/chemistryABSTRACT
Ursodeoxycholic acid, 250 to 300, 500 to 600, or 900 to 1000 mg/d, was given orally for 6 to 38 months to 53 patients with cholesterol gallstones and functioning gallbladders. Forty-two patients had greater than 50% reduction in gallstone volume, number, or both, without apparent dose dependence and 27 of these patients had complete gallstone dissolution. Results of laboratory studies including liver function tests were not affected adversely and biliary lithocholic acid concentration did not increase during therapy. Most biliary symptoms seemed to disappear within 3 months and no patient developed diarrhea. Large diameter and increased number of gallstones were found to hinder dissolution. The percentage of biliary ursodeoxycholic acid increased with increasing dose and reached a maximum of 50% to 60% of total bile acids at a dose of about 10 to 12 mg/kg body weight. d. Biliary lithogenic index was reduced significantly during treatment with ursodeoxycholic acid, 500 to 600 and 900 to 1000 mg/d. Thus, ursodeoxycholic acid appears to be a safe and effective alternative to surgery in selected patients with gallstones.
Subject(s)
Cholelithiasis/drug therapy , Deoxycholic Acid/analogs & derivatives , Ursodeoxycholic Acid/therapeutic use , Bile Acids and Salts/metabolism , Cholesterol/blood , Cholesterol/metabolism , Clinical Trials as Topic , Diarrhea/chemically induced , Dose-Response Relationship, Drug , Female , Humans , Lithocholic Acid/blood , Liver/pathology , Male , Middle Aged , Random Allocation , Time Factors , Ursodeoxycholic Acid/administration & dosage , Ursodeoxycholic Acid/adverse effectsABSTRACT
The effect of 7-ketolithocholic acid on biliary bile acid composition, cholesterol saturation, and as an intermediate in the conversion of chenodeoxycholic acid to ursodeoxycholic acid was investigated in 5 subjects with gallstones. After 7-ketolithocholic acid (400 mg/day) was administered orally for 14 days, biliary bile acid composition changed: The proportion of cholic acid decreased (from 45% to 19%), deoxycholic acid decreased (from 15% to 10%), chenodeoxycholic acid increased markedly (from 36% to 59%), ursodeoxycholic acid increased (from 36% to 59%), ursodeoxycholic acid increased (from 2% to 7%), and lithocholic acid increased (from 2% to 5%), while only trace amounts of 7-ketolithocholic acid were detected. During this treatment, the biliary lithogenic index fell from 2.6 to 0.9 and was accompanied by a pronounced drop in biliary cholesterol concentration. After biliary bile acid levels became constant [24-14C]chenodeoxycholic acid was given intravenously as a pulse-label, and the resultant biliary ursodeoxycholic acid and lithocholic acid specific activity curves showed a precursor--product relationship with chenodeoxycholic acid. Similarly, when uniformly labeled 7-[24-14C]ketolithocholic acid was fed (400 mg/day, 1000 +/- 100 dpm/mg) the specific activities of biliary chenodeoxycholic acid and ursodeoxycholic acid became constant and approximated each other, but these were only 75% as high as the fed 7-ketolithocholic acid. These results indicate that 7-ketolithocholic acid is absorbed, and suppresses endogenous bile acid production and biliary cholesterol secretion. Both isotopic experiments infer that ursodeoxycholic acid and lithocholic acid are formed from chenodeoxycholic acid and not from 7-ketolithocholic acid. The reduction in biliary lithogenic index and in cholesterol concentration suggest that low doses of 7-ketolithocholic acid may be effective in dissolving gallstones.
